RESUMO
<p><b>OBJECTIVE</b>To investigate the mechanism of clinical haemorrhage in an inherited coagulation factor VII (FVII) deficiency and tissue factor abnormality pedigree.</p><p><b>METHODS</b>All exons, exon-intron boundaries and the 3', 5' untranslated sequences of FVII and tissue factor (TF) genes were amplified by PCR and sequenced directly. Any mutation identified by direct sequencing was confirmed by reverse sequencing. FVII cDNA of the proband was synthesized with random primers and amplified by nest PCR.</p><p><b>RESULTS</b>55C-->T heterozygous mutation located in promoter of FVII gene was identified in the proband. The heterozygous mutation was derived from his mother. Tracing the other pedigree members found that his sister had the same heterozygous mutation and the others had wild-type FVII genes. A 9363 C-->T (Arg131Trp) heterozygous polymorphism in TF gene, which was 2.63% frequency of T allele polymorphism, was found in all of the pedigree members.</p><p><b>CONCLUSION</b>It was the first report that the -55C-->T heterozygous mutation in FVII gene and the Arg131Trp heterozygous polymorphism in TF gene explained the clinical symptom of the proband.</p>
Assuntos
Adulto , Humanos , Masculino , Análise Mutacional de DNA , Fator VII , Genética , Deficiência do Fator VII , Genética , Heterozigoto , Linhagem , Polimorfismo Genético , Tromboplastina , GenéticaRESUMO
<p><b>OBJECTIVE</b>To study the molecular mechanism of antithrombin (AT) gene C2759T (Leu99Phe) mutation causing AT deficiency.</p><p><b>METHODS</b>A mutated AT cDNA expression plasmid ATM2759 was constructed by mega-primer method. ATM2759 and wild type AT cDNA expression plasmid ATN were transfected into COS7 cells or CHO cells by using Superfect reagent respectively for in vitro expression study and immunofluorescence assay.</p><p><b>RESULTS</b>The antigen levels of AT (AT:Ag) in the cell lysate of ATM2759 transfected COS7 cells and the cell culture supernatant were 174.97% and 35.63% of that of ATN transfected COS7 cells respectively, whereas the AT activity in the cell culture supernatant was 47.73% of the control's. Immunofluorescence analysis showed that the fluorescence intensity was significantly higher in ATM2759 transfected CHO cells than in those transfected with ATN.</p><p><b>CONCLUSIONS</b>Leu99Phe substitution may not affect the binding capacity of AT with heparin. Secretion defect and intracellular accumulation of the mutated AT protein might be the mechanisms of this mutation causing AT deficiency.</p>
Assuntos
Animais , Cricetinae , Antitrombina III , Genética , Metabolismo , Deficiência de Antitrombina III , Genética , Células CHO , Células COS , Chlorocebus aethiops , Cricetulus , Imunofluorescência , Mutação , Plasmídeos , Genética , TransfecçãoRESUMO
<p><b>OBJECTIVE</b>To identify gene defect in a Chinese pedigree of hereditary coagulation factor XI (FXI) deficiency.</p><p><b>METHODS</b>The peripheral blood samples were collected from the proband and her family members. The plasma PT, APTT, FXI:C and FXI:Ag were assayed. The FXI gene exons and exon-intron boundaries of the proband were amplified by PCR and then sequenced directly. The mRNA of FXI in the peripheral blood was analyzed with RT-PCR.</p><p><b>RESULTS</b>The proband and some of her family members had prolonged APTT. The plasma FXI:C and FXI:Ag of the proband, her brother and her parents were lower than 10% and 50% of the normal values, respectively. Nucleotide sequence analysis revealed that the proband and her brother had a homozygous mutation of IVS J-4delgttg in FXI gene. The mutation was inherited from her parents who were heterozygotes. The mutation was not found in 60 normal subjects. No FXI mRNA was detected in peripheral blood sample of the proband.</p><p><b>CONCLUSION</b>The IVS J-4delgttg is a novel mutation causing FXI deficiency, which may interfere with mRNA splicing.</p>
Assuntos
Adulto , Feminino , Humanos , Sequência de Bases , Análise Mutacional de DNA , Fator XI , Genética , Deficiência do Fator XI , Sangue , Genética , Patologia , Genótipo , Íntrons , Genética , Dados de Sequência Molecular , Tempo de Tromboplastina Parcial , Linhagem , Fenótipo , Mutação Puntual , Tempo de Protrombina , RNA Mensageiro , Genética , Metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Deleção de SequênciaRESUMO
<p><b>OBJECTIVE</b>To identify gene mutations of a pedigree with inherited factor V (FV) deficiency.</p><p><b>METHODS</b>The activated partial thromboplastin time (APTT), prothrombin time (PT), FV activity (FV:C) and FV antigen (FV:Ag) tests were performed for phenotypic diagnosis. The genomic DNA was extracted from the peripheral blood of the proband and all the 25 exons and their flanks of FV gene were amplified by polymerase chain reaction (PCR). The PCR products were screened by direct sequencing and the mutations were further confirmed by restriction enzyme digestion.</p><p><b>RESULTS</b>APTT, PT, TT, FV:C, FV:Ag of the proband were 249.2 s, 46.6 s, 17.9 s, 0.1% and 1.5%, respectively. FII, FVII, FVIII, FIX, FX activities, vWF and Fg were within normal ranges. Taking the GenBank Z99572 sequence as the reference, four mutations were identified in FV gene of the proband. They were a heterozygous two bases deletion in exon 13 (2238 approximately 2239delAG) introducing a frameshift and a premature stop at codon 689, and a heterozygous missense mutation in exon 23 (G6410T) resulting in the substitution of Gly for Val at codon 2079, respectively. The proband's father and mother were heterozygous for G6410T and for 2238 approximately 2239delAG, respectively.</p><p><b>CONCLUSION</b>The severe FV deficiency of the proband is caused by a frameshift mutation of 2238 approximately 2239delAG and a missense mutation of G6410T, which haven't been identified before.</p>
Assuntos
Adulto , Feminino , Humanos , Lactente , Masculino , Sequência de Bases , Análise Mutacional de DNA , Éxons , Genética , Fator V , Genética , Metabolismo , Deficiência do Fator V , Genética , Mutação da Fase de Leitura , Heterozigoto , Mutação de Sentido Incorreto , Tempo de Tromboplastina Parcial , Linhagem , Fenótipo , Tempo de Protrombina , Tempo de TrombinaRESUMO
<p><b>OBJECTIVE</b>To investigate the pathogenesis of inherited coagulation factor VII (FVII) deficiency.</p><p><b>METHODS</b>The diagnosis was validated by coagulant parameter assay. FVII gene mutations were analysed in the proband by DNA direct sequencing of PCR products of all exons, exon-intron boundaries and the 3', 5'untranslated sequences. The mutations were confirmed by reverse sequencing. The ectopic transcripts of RT-PCR were used to confirm the characteristics of the mutation in non-canonical splice site (IVS1a + 5g > a).</p><p><b>RESULTS</b>Double heterozygous mutations in the propositus were identified: a T to G mutation at position 10961, resulting in His348Gln substitution, a non-canonical splice site (IVS1a + 5g > a) mutation, causing the new model of splice and frameshift mutation.</p><p><b>CONCLUSION</b>Double heterozygous mutations of His348Gln and IVS1a + 5g > a were identified in a propositus, the splicing pattern of the IVS1a + 5g > a mutation was reported for the first time.</p>
Assuntos
Humanos , Sequência de Aminoácidos , Sequência de Bases , Fator VII , Genética , Deficiência do Fator VII , Genética , Heterozigoto , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Splicing de RNARESUMO
<p><b>OBJECTIVES</b>To identify the FXI gene mutations in two Chinese pedigrees of congenital factor XI deficiency.</p><p><b>METHODS</b>The peripheral blood samples were collected from the probands and their family members and the plasma FXI:C and FXI:Ag were determined. All the exons and exon-intron boundries of FXI gene were amplified with PCR and sequenced thereafter.</p><p><b>RESULTS</b>A nonsense mutation Trp228stop and two missense mutations Glu323Lys and Leu172Pro were disclosed in the two pedigrees. All mutations existed in a heterozygous state.</p><p><b>CONCLUSION</b>The FXI gene mutations Trp228stop, Glu323Lys and Leu172Pro attribute to the pathogenesis of the congenital factor XI deficiency in Chinese. The Leu172Pro is identified for the first time.</p>
Assuntos
Adulto , Criança , Humanos , Masculino , Pessoa de Meia-Idade , Povo Asiático , Genética , Sequência de Bases , Fator XI , Genética , Deficiência do Fator XI , Genética , Dados de Sequência Molecular , Mutação , LinhagemRESUMO
The aim is to observe the expression of human factor VIII gene in mice tranduced in vivo and ex vivo. The vector pLNC-FVIII BD was generated by cloning a B-domain-deleted (760aa-1639aa) FVIII cDNA (FVIIIBD cDNA) into retroviral vector pLNCX. 2 x 10(6) of mouse bone marrow stroma cells transduced by LNC-FVIII BD were infused into 4-week-old BALB/c mice by tail-vein injection. pLNC-FVIII BD was conjugated with PAMAM dendrimer to form complex PAMAM-pLNC-FVIII BD, with which C57BL/6J were injected by tail vein (200 micro l contained 15 micro g/mouse) and sacrificed at days 1, 2, 7, 14, 21 and 28, respectively after injection. Tissue such as liver, spleen, lung and kindney were harvested, with which the transcription were detected by means of RT-PCR. In addition, blood was collected to be measured human FVIII Ag, human FVIIIc and anti-FVIII of human inhibitors. The results showed that the highest level of human FVIII in the recipient BALB/c mice was 8.6 +/- 1.44 ng/ml detected on the first day post-injection; anti-FVIII antibodies were detected from the first week post-injection, and then the level of FVIII Ag decreased and cannot be measured on the fourth week. In the C57BL/6J mice physiological level of human FVIII was expressed in plasma at 48 hours after injection and the average human FVIIIc was 0.62 U/ml and the average human FVIII Ag was 115.5 ng/ml, and gradually reduced later. Anti-FVIII of human inhibitors was not revealed all the time. Syngene image scanning demonstrated that the transcription of the human FVIII BD cDNA occurred mainly in spleen and lung, and secondarily in liver and kidney. No side effects of PAMAM-pLNC-FVIII BD were observed in mice tissue by pathological examination at 4 weeks. In conclusion, retrovirus-transduced bone marrow stroma cells effectively produced human FVIII after ex vivo transduction, but the development of anti-FVIII antibodies in recipient mice influenced the expression level. The human FVIII gene can successfully be transduced in vivo through injecting PAMAM-pLNC-FVIII BD cDNA into mice intravenously. There was physiological level expression of human FVIII in plasma at 48 hours after injection and the average human FVIIIc is 0.62 U/ml and the peak in the six mice was 0.89 U/ml, and gradually reduced later.
Assuntos
Animais , Camundongos , DNA Complementar , Fator VIII , Genética , Terapia Genética , Hemofilia A , Terapêutica , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , TransfecçãoRESUMO
To investigate the non-viral vector mediating human coagulation factor VIII gene expression in mouse 32D cell line, a recombinant plasmid vector, pRC/RSV-hFVIIIBDcDNA, was constructed by cloning B-domain-deleted (Delta760aa-1639aa) human factor VIII cDNA (hFVIIIBDcDNA) into plasmid vector, pRC/RSV. The plasmid RC/RSV-hFVIIIBDcDNA was then transfected by means of SuperFect Transfection Reagent into mouse 32D cell line. After screening with G418, the procoagulant activity (hFVIII:C) and antigen (hFVIII:Ag) of human factor VIII in the culture medium were detected using one-stage method and ELISA, respectively. Furthermore, RT-PCR was performed to observe the transcription of hFVIIIBDcDNA. The results showed that human coagulation factor VIII protein existed in culture medium with hFVIII:C up to 2.01 U/(10(6) cell x 24 hours) and hFVIII:Ag to 450.08 ng/(10(6) cell x 24 hours). RT-PCR displayed mRNA of hFVIIIBDcDNA in 32D cells. It is concluded that the recombinant plasmid RC/RSV-hFVIIIBDcDNA can successfully express human FVIII in mouse 32D cell line, and hFVIII expressed in vitro presents the similar coagulant activity to the native hFVIII existing in normal human plasma.
Assuntos
Animais , Humanos , Camundongos , Linhagem Celular , DNA Complementar , Genética , Ensaio de Imunoadsorção Enzimática , Fator VIII , Genética , Metabolismo , Regulação da Expressão Gênica , Plasmídeos , Genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
<p><b>OBJECTIVE</b>To identify the gene mutations in a pedigree with hereditary hemorrhagic telangiectasia.</p><p><b>METHODS</b>Genomic DNA was extracted from the peripheral blood of the propositus. All of the exons, intron/exon boundaries and the 5' untranslation regions (UTR) of the ALK-1 and endoglin gene were amplified by polymerase chain reaction (PCR). The PCR products were screened by direct sequencing.</p><p><b>RESULTS</b>The mutation is a C1437T substitution in exon 10 of the ALK-1 gene, resulting in Arg 479 Stop.</p><p><b>CONCLUSION</b>The hereditary hemorrhagic telangiectasia propositus is caused by a heterozygous Arg 479 Stop mutation in the ALK-1 gene which has not been identified previously.</p>
Assuntos
Idoso , Feminino , Humanos , Masculino , Receptores de Activinas Tipo II , Genética , Antígenos CD , Genética , Sequência de Bases , Códon sem Sentido , Análise Mutacional de DNA , Éxons , Genética , Linhagem , Mutação Puntual , Receptores de Superfície Celular , Genética , Telangiectasia Hemorrágica Hereditária , Genética , PatologiaRESUMO
<p><b>OBJECTIVE</b>To demonstrate the effectiveness of a retrovirus-based plasmid vector coupled with nanometer material-polyamidoamine (PAMAM) dendrimer in stable gene expression of FVIII in vitro and to study the cytotoxicity of PAMAM.</p><p><b>METHODS</b>The retrovirus-based plasmid vector pLNC-FVIII BD was generated by cloning a B-domain-deleted (760aa - 1639aa) FVIII cDNA (FVIIIBD cDNA) into retroviral vector pLNCX. The complex that contained PAMAM and pLNC-FVIII BD transfer FVIII BD cDNA into NIH3T3 cell line. In day 2, 5, 10, 15, 30 after transferring, the antigen and procoagulant activity of human FVIII in the cell culture medium were measured by ELISA assay and one-stage method, respectively. RT-PCR was performed for the detection of FVIII BD mRNA. Inhibitory percentage of cell vitality was used for cytotoxicity of PAMAM.</p><p><b>RESULTS</b>Human FVIII was expressed for 30 days by transfected cells. The mean procoagulant activity of secreted FVIII in these 30 days was 0.929 U/ml, and the FVIII antigen was 0.188 micro g/ml by 10(6) cells in 24 hours, respectively. The level of FVIII didn't significantly decreased during these days. Inhibitory percent of cell vitality was only 5.32%.</p><p><b>CONCLUSION</b>PAMAM could effectively transfer pLNC-FVIII BD into NIH3T3 cells and FVIII could be stably and effectively expressed by the transfected cells. Cytotoxicity of PAMAM was low.</p>
Assuntos
Animais , Camundongos , Dendrímeros , Fator VIII , Genética , Vetores Genéticos , Genética , Células NIH 3T3 , Plasmídeos , Poliaminas , Farmacologia , Retroviridae , GenéticaRESUMO
<p><b>OBJECTIVE</b>To investigate the gene mutations in a pedigree with inherited prothrombin (FII) deficiency.</p><p><b>METHODS</b>The activated partial thromboplastin time (APTT), prothrombin time (PT), FII activity (FII:C) and FII antigen (FII:Ag) test were used for phenotype diagnosis. The genomic DNA was extracted from the peripheral blood of the propositus. All the 14 exons, intron/exon boundaries and the 5' and 3' untranslated regions (UTR) of the prothrombin gene were amplified by polymerase chain reaction (PCR). The PCR products were screened by direct sequencing and the mutations detected were further confirmed by restricted enzyme digestion. One hundred and three healthy blood donors were used as controls.</p><p><b>RESULTS</b>The phenotype of the propositus was prothrombin deficiency (type I). With reference to the prothrombin nucleotide sequence published by Degen & Dacie, three variations were found in the FII gene of the propositus. Among them, the novel mutation was a homozygous A601G subtitution in exon 2.</p><p><b>CONCLUSION</b>The prothrombin deficiency of the propositus is caused by a homozygous Glu29 to Gly mutation in the prothrombin gene.</p>
Assuntos
Criança , Feminino , Humanos , Coagulação Sanguínea , Hipoprotrombinemias , Sangue , Genética , Mutação Puntual , Protrombina , GenéticaRESUMO
<p><b>OBJECTIVE</b>To explore the gene mutation type of an inherited coagulation factor VII deficiency pedigree.</p><p><b>METHODS</b>FVII:Ag, FVII:C, FVIIa were detected to classify deficiency type. FVII gene mutations were analysed in the proband and her family members by DNA directly sequencing. Biostructural pathology of the identified mutation was analysed by molecular modeling.</p><p><b>RESULTS</b>Homozygosity of C-->T transition at position 11514 in exon 8 resulting in Thr359Met was identified in the proband, and heterozygosity for Thr359Met was confirmed in her parents, her son and some other family members. Thr359Met induces CRM-deficiency. It is found by computer simulated molecular model that the replacement of Thr by Met which has a larger and longer side chain might cause steric hindrance, and change the number of H-bonds.</p><p><b>CONCLUSIONS</b>Homozygous missense mutation Thr359Met was found in a pedigree of hereditary FVII deficiency. This mutation might change the configuration of protein molecule and result in severe FVII deficiency.</p>
Assuntos
Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise Mutacional de DNA , Fator VII , Genética , Deficiência do Fator VII , Genética , Homozigoto , Mutação de Sentido Incorreto , Linhagem , Reação em Cadeia da PolimeraseRESUMO
<p><b>OBJECTIVE</b>To identify the factor XI gene mutation in a Chinese pedigree of congenital factor XI deficiency.</p><p><b>METHODS</b>The peripheral blood samples were collected from the proband and her family members and the plasma FXI:C and FXI:Ag were assayed. All the exons and their adjacent intron sequences of factor XI were amplified with PCR and sequenced thereafter.</p><p><b>RESULTS</b>Two novel nonsense mutations TGG-->TGA (Trp228stop) and TGG-->TAG (Trp383stop) were identified in the family.</p><p><b>CONCLUSION</b>The compound heterozygous Trp228stop and Trp383stop may attribute to the pathogenesis of the congenital factor deficiency.</p>
Assuntos
Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Povo Asiático , Códon sem Sentido , Fator XI , Genética , Deficiência do Fator XI , Genética , Linhagem , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
<p><b>OBJECTIVE</b>To investigate the genotypes of mutations of an inherited coagulation factor VII(F VII) deficiency pedigree.</p><p><b>METHODS</b>The diagnosis was validated by coagulant parameters. F VII gene mutations were analysed in the proband and her family members by DNA direct sequencing. The PCR fragments were cleaved by the Msp I restriction enzyme to confirm the mutations detected by sequencing was performed in this study.</p><p><b>RESULTS</b>Double heterozygous mutations at the same coding site of amino acid were detected in propositus of the pedigree: a C to T mutation at position 11348 resulting in Arg304Trp substitution combined with a G to A mutation at position 11349 resulting in Arg304Gln substitution. Her farther had a G to A mutation at position 11349 and her mother had a C to T mutation at position 11348, respectively. Both were heterozygous mutations. One of her brothers had normal genotype, the other brother and all her three offsprings had heterozygous mutations.</p><p><b>CONCLUSION</b>Double heterozygous mutations coding the same amino acid were found in a pedigree with hereditary coagulation factor VII deficiency.</p>